The presence of magnet increases the extent of ligation in bead-bead subcloning.

<p>Flow cytometry of fluorescent-labeled beads allows quantification of bead-bead ligations. Insert beads are red-labeled (Alexa488 label) and acceptor-vector beads are green-labeled (Alexa647 label). Successful bead-bead ligations appear at high FL-1 and FL-6 readouts (boxed). The insert is ITGA2b (1 350 bp) and the acceptor vector is pLenti1 (8 180 bp). (A) Ligation in the absence of magnet. Approximately 0.5% of acceptor beads are ligated. (B) Ligation in the presence of magnet. Approximately 7% of the acceptor beads are ligated, a 14-fold increase in extent of ligation.</p

Ligation of solution phase DNA to a bead-immobilized vector.

<p>(<b>A</b>) A fluorescence-based assay for determining extent of ligation. Beads with immobilized vector are incubated with a Alexa 488 fluorescent oligo. The extent of ligation is measured via flow cytometry. Bead loading was at 1 ng vector DNA (pHISZ)/ug bead vector. Positive: Beads in which pHISZ vector is fully fluorescently labeled. Ligation: Beads after ligation. Negative: Beads in which pHISZ vector is not fluorescently labeled. The extent of ligation f is measured as a percentage of the Positive signal. (<b>B</b>) Extent of ligation is reduced at high bead loadings.</p

Bead-bead subcloning of cancer genes into multiple expression vectors.

<p>The ectodomains of three cancer antigens (TNF, CA9 ectodomain, and PSMA ectodomain) were subcloned via bead-bead subcloning from an <i>E. coli</i> expression vector into expression vectors for <i>S. carnosus, P. pastoris</i>, and CHO. (A) PCR screens of pLentiHAp transformants show correct sizes for 10/10. (B) Expression of target proteins on the surface of <i>S. carnosus</i> using the surface display vector pSCEM2. The surface expression is quantified via flow cytometry as HSA-Alexa647 binding to ABP, which is co-expressed with the target protein. (C) Expression of target proteins from the CHO expression vector pLenitHAp. Western blotting (anti His6) of CHO cell lysates. (D) Expression of target proteins from the <i>P. pastoris</i> expression vector pPICZap. Western blotting (anti His6) of <i>P. pastoris</i> cell lysates.</p

Statistics of automated bead-bead subcloning of 95 target genes.

<p>Statistics of automated bead-bead subcloning of 95 target genes.</p

Ligation efficiencies of bead-based subcloning strategies.^*

*<p> <i>Vector and donor beads were loaded at 0.5 ng DNA/µg bead.</i></p